Álvaro Martínez-del-Pozo

The behavior of sea anemone actinoporins at the water-membrane interface.

Actinoporins constitute a group of small and basic α-pore forming toxins produced by sea anemones. They display high sequence identity and appear as multigene families. They show a singular behaviour at the water-membrane interface: In aqueous solution, actinoporins remain stably folded but, upon interaction with lipid bilayers, become integral membrane structures. These membranes contain sphingomyelin, display phase coexistence, or both. The water soluble structures of the actinoporins equinatoxin II (EqtII) and sticholysin II (StnII) are known in detail. The crystalline structure of a fragaceatoxin C (FraC) nonamer has been also determined. The three proteins fold as a β-sandwich motif flanked by two α-helices, one of them at the N-terminal end. Four regions seem to be especially important: A cluster of aromatic residues, a phosphocholine binding site, an array of basic amino acids, and the N-terminal α-helix. Initial binding of the soluble monomers to the membrane is accomplished by the cluster of aromatic amino acids, the array of basic residues, and the phosphocholine binding site. Then, the N-terminal α-helix detaches from the β-sandwich, extends, and lies parallel to the membrane. Simultaneously, oligomerization occurs. Finally, the extended N-terminal α-helix penetrates the membrane to build a toroidal pore. This model has been however recently challenged by the cryo-EM reconstruction of FraC bound to phospholipid vesicles. Actinoporins structural fold appears across all eukaryotic kingdoms in other functionally unrelated proteins. Many of these proteins neither bind to lipid membranes nor induce cell lysis. Finally, studies focusing on the therapeutic potential of actinoporins also abound.

Calorimetric Scrutiny of Lipid Binding by Sticholysin II Toxin Mutants

The mechanisms by which pore-forming toxins are able to insert into lipid membranes are a subject of the highest interest in the field of lipid-protein interaction. Eight mutants affecting different regions of sticholysin II, a member of the pore-forming actinoporin family, have been produced, and their hemolytic and lipid-binding properties were compared to those of the wild-type protein. A thermodynamic approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments show that pore formation by sticholysin II is an enthalpy-driven process that occurs with a high affinity constant (1.7x10(8) M(-1)). Results suggest that conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, although it is necessary for correct pore formation. Membrane binding is achieved through two separate mechanisms, that is, recognition of the lipid-water interface by a cluster of aromatic residues and additional specific interactions that include a phosphocholine-binding site. Thermodynamic parameters derived from titration experiments are discussed in terms of a putative model for pore formation.

A PR-1-like Protein of Fusarium oxysporum Functions in Virulence on Mammalian Hosts

Background: Pathogenesis-related (PR-1-like) proteins are widely conserved in eukaryotes, but their biological function is unknown. Results: Knockout or site-directed mutagenesis of fpr1 encoding a secreted PR-1-like protein in the fungal pathogen Fusarium oxysporum impairs virulence on mice. Conclusion: Secreted PR-1-like proteins are important for fungal infection of mammals. Significance: We show the first genetic evidence for a biological function of the predicted active site of PR-1-like proteins. The pathogenesis-related PR-1-like protein family comprises secreted proteins from the animal, plant, and fungal kingdoms whose biological function remains poorly understood. Here we have characterized a PR-1-like protein, Fpr1, from Fusarium oxysporum, an ubiquitous fungal pathogen that causes vascular wilt disease on a wide range of plant species and can produce life-threatening infections in immunocompromised humans. Fpr1 is secreted and proteolytically processed by the fungus. The fpr1 gene is required for virulence in a disseminated immunodepressed mouse model, and its function depends on the integrity of the proposed active site of PR-1-like proteins. Fpr1 belongs to a gene family that has expanded in plant pathogenic Sordariomycetes. These results suggest that secreted PR-1-like proteins play important roles in fungal pathogenicity.

Specific interactions of sticholysin I with model membranes: An NMR study

Sticholysin I (StnI) is an actinoporin produced by the sea anemone Stichodactyla helianthus that binds biological and model membranes forming oligomeric pores. Both a surface cluster of aromatic rings and the N‐terminal region are involved in pore formation. To characterize the membrane binding by StnI, we have studied by 1H‐NMR the environment of these regions in water and in the presence of membrane‐mimicking micelles. Unlike other peptides from homologous actinoporins, the synthetic peptide corresponding to residues 1–30 tends to form helix in water and is more helical in either trifluoroethanol or dodecylphosphocholine (DPC) micelles. In these environments, it forms a helix‐turn‐helix motif with the last α‐helical segment matching the native helix‐α1 (residues 14–24) present in the complete protein. The first helix (residues 4–9) is less populated and is not present in the water‐soluble protein structure. The characterization of wild‐type StnI structure in micelles shows that the helix‐α1 is maintained in its native structure and that this micellar environment does not provoke its detachment from the protein core. Finally, the study of the aromatic resonances has shown that the motional flexibility of specific rings is perturbed in the presence of micelles. On these bases, the implication of the aromatic rings of Trp‐111, Tyr‐112, Trp‐115, Tyr‐132, Tyr‐136, and Tyr‐137, in the interaction between StnI and the micelle is discussed. Based on all the findings, a revised model for StnI interaction with membranes is proposed, which accounts for differences in its behavior as compared with other highly homologous sticholysins. Proteins 2010.

2NH and 3OH are crucial structural requirements in sphingomyelin for sticholysin II binding and pore formation in bilayer membranes

Sticholysin II (StnII) is a pore-forming toxin from the sea anemone Stichodactyla heliantus which belongs to the large actinoporin family. The toxin binds to sphingomyelin (SM) containing membranes, and shows high binding specificity for this lipid. In this study, we have examined the role of the hydrogen bonding groups of the SM long-chain base (i.e., the 2NH and the 3OH) for StnII recognition. We prepared methylated SM-analogs which had reduced hydrogen bonding capability from 2NH and 3OH. Both surface plasmon resonance experiments, and isothermal titration calorimetry measurements indicated that StnII failed to bind to bilayers containing methylated SM-analogs, whereas clear binding was seen to SM-containing bilayers. StnII also failed to induce calcein release (i.e., pore formation) from vesicles made to contain methylated SM-analogs, but readily induced calcein release from SM-containing vesicles. Molecular modeling of SM docked to the phosphocholine binding site of StnII indicated that the 2NH and 3OH groups were likely to form a hydrogen bond with Tyr135. In addition, it appeared that Tyr111 and Tyr136 could donate hydrogen bonds to phosphate oxygen, thus stabilizing SM binding to the toxin. We conclude that the interfacial hydrogen bonding properties of SM, in addition to the phosphocholine head group, are crucial for high-affinity SM/StnII-interaction.

Three-dimensional structure of the actinoporin sticholysin I. Influence of long-distance effects on protein function

Actinoporins are water-soluble proteins with the ability to form pores upon insertion into biological membranes. They constitute a family of proteins with high degree of sequence identities but different hemolytic activities, suggesting that minor conformational arrangements result in major functional changes. A good example of this situation is the sea anemone Stichodactyla helianthus which produces two very similar actinoporins, sticholysins I (StnI) and II (StnII), but of very different hemolytic efficiency. Within this idea, given that the high resolution three-dimensional structure of StnII is already known, we have now solved that one corresponding to StnI in order to analyze the influence of particular residues on the conformation and activity of these proteins. In addition, random mutagenesis has been also used to produce five less hemolytic variants of StnI. All these mutations map to functionally relevant regions because they are probably involved in conformational changes associated with pore formation, which take place after membrane binding, and involve long-distance rearrangements of the polypeptide chain of actinoporins.

Intrinsic local disorder and a network of charge-charge interactions are key to actinoporin membrane disruption and cytotoxicity

Actinoporins are a family of sea anemone proteins that bind to membranes and produce functional pores which result in cell lysis. Actinoporin variants with decreased lytic activity usually show a reduced affinity for membranes. However, for some of these mutant versions there is no direct correlation between the loss of binding affinity and the decrease in their overall lytic activity, suggesting that other steps in pore formation may be hampered or facilitated by the mutations. To test this hypothesis on the mechanism of pore formation by this interesting family of proteins, structural and dynamic NMR studies have been carried out on two disabled variants of the actinoporin Sticholysin II, R29Q and Y111N. It is shown that their lytic activity is not only related to their membrane affinity but also to their conformational mechanism for membrane insertion. Alterations in their activities can be explained by structural, electrostatic and dynamic differences in a cluster of aromatic moieties and the N‐terminus. In addition, the dynamic properties of some segments located at the C‐terminus of the R29Q variant suggest a relevant role for this region in terms of protein–protein interactions. On the basis of all these results, we propose that R29 anchors a network of electrostatic interactions crucial for the actinoporin’s approach to the membrane and that Y111 induces a necessary disorder in the loop regions that bind to membranes.

Cholesterol stimulates and ceramide inhibits Sticholysin II-induced pore formation in complex bilayer membranes

The pore forming capacity of Sticholysin II (StnII; isolated from Stichodactyla helianthus) in bilayer membranes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), palmitoylsphingomyelin (PSM) and either cholesterol or palmitoyl ceramide (PCer) has been examined. The aim of the study was to elucidate how the presence of differently ordered PSM domains affected StnII oligomerization and pore formation. Cholesterol is known to enhance pore formation by StnII, and our results confirmed this and provide kinetic information for the process. The effect of cholesterol on bilayer permeabilization kinetics was concentration-dependent. In the concentration regime used (2.5-10nmol cholesterol in POPC:PSM 80:20 by nmol), cholesterol also increased the acyl chain order in the fluid PSM domain and thus decreased bilayer fluidity, suggesting that fluidity per se was not responsible for cholesterols effect. Addition of PCer (2.5-10nmol) to the POPC:PSM (80:20 by nmol) bilayers attenuated StnII-induced pore formation, again in a concentration-dependent fashion. This addition also led to the formation of a PCer-rich gel phase. Addition of cholesterol to PCer-containing membranes could partially reduce the inhibitory effect of PCer on StnII pore formation. We conclude that the physical state of PSM (as influenced by either cholesterol or PCer) affected StnII binding and pore formation under the conditions examined.

Production of the biotechnologically relevant AFP from Aspergillus giganteus in the yeast Pichia pastoris

The mould Aspergillus giganteus produces a basic, low molecular weight protein (AFP) showing in vitro and in vivo antifungal properties against important plant pathogens. AFP is secreted as an inactive precursor containing an amino-terminal extension of six amino acids (lf-AFP) which is later removed to produce the active protein. The molecular basis to explain this behavior and the features that determine the fungal specificity of this protein are not completely solved. In this work, the mature AFP (AFP *) and a version of AFP with an extended amino-terminal (proAFP) have been cloned and produced in the yeast Pichia pastoris. The two proteins have been purified to homogeneity and characterized from structural and functional points of view. Recombinant AFP * produced is practically indistinguishable from the natural fungal protein in terms of its spectroscopic and antifungal properties while proAFP is mostly inactive under identical assay conditions. The availability of an active AFP protein produced in P. pastoris will permit investigation of the mode of action and targeting specificity of AFP by using site-directed mutagenesis approaches.

Production and characterization of a colon cancer-specific immunotoxin based on the fungal ribotoxin α-sarcin

A single-chain fusion protein that directed the cytolytic activity of α-sarcin to A33 tumor antigen expressing cells was constructed and shown to effectively kill targeted cells. Glycoprotein A33 (GPA33) is a well-known colon cancer marker and a humanized antibody against it was used to target the α-sarcin. The fungal ribotoxin α-sarcin is one of the most potent and specific toxins known. It is small, protease resistant, thermostable and highly efficient towards the inactivation of ribosomes. This work describes the production and characterization of an immunotoxin resulting from fusing the single-chain variable fragment (scFv) of the monoclonal antibody that targets GPA33 to fungal α-sarcin. This chimeric protein (scFvA33αsarcin), produced in Pichia pastoris and purified in high yield was proven to be properly folded, active, specific and stable. It showed high specific toxicity against GPA33-positive tumoral cell lines providing scientific evidence to sustain that scFvA33αsarcin is a good immunotherapeutic candidate against GPA33-positive colon carcinomas.